Biosynthesis is an environmentally benign and renewable approach that can be used to produce a broad range of natural and, in some cases, new-to-nature products. However, biology lacks many of the reactions that are available to synthetic chemists, resulting in a narrower scope of accessible products when using biosynthesis rather than synthetic chemistry. A prime example of such chemistry is carbene-transfer reactions1. Although it was recently shown that carbene-transfer reactions can be performed in a cell and used for biosynthesis2,3, carbene donors and unnatural cofactors needed to be added exogenously and transported into cells to effect the desired reactions, precluding cost-effective scale-up of the biosynthesis process with these reactions. Here we report the access to a diazo ester carbene precursor by cellular metabolism and a microbial platform for introducing unnatural carbene-transfer reactions into biosynthesis. The α-diazoester azaserine was produced by expressing a biosynthetic gene cluster in Streptomyces albus. The intracellularly produced azaserine was used as a carbene donor to cyclopropanate another intracellularly produced molecule—styrene. The reaction was catalysed by engineered P450 mutants containing a native cofactor with excellent diastereoselectivity and a moderate yield. Our study establishes a scalable, microbial platform for conducting intracellular abiological carbene-transfer reactions to functionalize a range of natural and new-to-nature products and expands the scope of organic products that can be produced by cellular metabolism.
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The DNA sequences of plasmids used in this study have been deposited in the public version of the JBEI registry (http://public-registry.jbei.org). Accession codes are provided in Supplementary Table 2 (Part ID). The sequences and annotation of the aza BGC is available at GenBank (NZ_BEVZ01000003.1) spanning bases 82400–111549. The determined structure has been deposited in the PDB (8FBC). The MS proteomics data have been deposited at the ProteomeXchange Consortium through the PRIDE63 partner repository under the dataset identifier PXD037509. Source data are provided with this paper.
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This work was supported by Joint BioEnergy Institute (https://www.jbei.org), which is supported by the DOE, Office of Science, Office of Biological and Environmental Research under contract DE-AC02-05CH11231 and National Science Foundation grant 2027943. We thank H. Celik and the staff at UC Berkeley’s NMR facility in the College of Chemistry (CoC-NMR) for spectroscopy assistance. Instruments in the CoC-NMR are supported in part by NIH S10OD024998.
J.D.K. has a financial interest in Amyris, Demetrix, Maple Bio, Lygos, Napigen, Berkeley Yeast, Zero Acre Farms, Ansa Biotechnologies, Apertor Pharmaceuticals, ResVit Bio and Cyklos Materials. The other authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 Effect of Na2S2O4 on the activity of haemin and Ir(Me)MPIX for the reaction of styrene with azaserine.
a, Addition of Na2S2O4 decreased the reaction yield when using Ir(Me)MPIX as catalyst. TON, turn-over number. Reaction conditions are described in Fig. 2 legend. Data are mean value for 2 reaction replicates. b, Na2S2O4 is necessary for the activity of haemin toward the reaction. EIC ([M+H]+, m/z 250.1074) for target products. The traces are representative of two reaction replicates. The reaction contained 5 mM styrene, 5 mM azaserine, 10 µM haemin or no catalyst, 0 or 10 mM Na2S2O4, 5 vol% ethanol, and M9-N buffer and was conducted at 22 °C under aerobic conditions for 18 h. Standard, chemically synthesized authentic standard mixture of the four diastereomers.
Extended Data Fig. 2 Azaserine toxicity on E. coli and S. albus.
a, Azaserine is toxic to E. coli BL21(DE3). b, Azaserine does not affect the growth of S. albus under tested concentrations. Biomass was normalized to that of culture without addition of azaserine. Data are mean ± s.d.; n = 3 biological replicates.
Extended Data Fig. 3 Expression of azaserine gene cluster from S. fragilis in S. albus.
a, Bioinformatic annotation of the azaserine gene cluster and comparison with the biosynthetic gene clusters for some natural N–N bond-containing compounds. b, Proteomic analysis of the azaserine gene cluster when expressed in S. albus. Data are mean ± s.d.; n = 3 biological replicates.
Extended Data Fig. 4 Differences in azaserine degradation in various media.
a, Azaserine titre continues to decrease after removal of S. albus cells. The azaserine-producing S. albus was grown in TSB medium. After 24 h, the cells were removed from culture broth using a 0.22-µm sterile filter, the filtrate was incubated at 30 °C (labelled as 0 h), and the azaserine concentration was monitored at different time points. b, Azaserine is stable in fresh TSB medium of normal pH 7.3 or adjusted pH 8.4. Equal volume of azaserine stock was added to a final concentration of about 35 mg/L at 0 h. Data are mean ± s.d.; n = 3 biological (a) or technical (b) replicates.
Extended Data Fig. 5 Purified P450-T2 WT and mutant catalysing the reaction of styrene with azaserine in vitro.
a, Coomassie Blue stained SDS-PAGE gel of purified protein P450-T2 WT (left) and P450-T2-5 mutant (right). Each lane is a sample from fractions collected during ion exchange purification. b, Purified P450-T2 WT and P450-T2-5 mutant proteins catalyse the reaction in vitro with high diastereoselectivity. 5th, P450-T2-5 mutant. Reaction conditions: 5 mM styrene, 5 mM azaserine, 10 µM enzyme, 10 mM Na2S2O4, 5 vol% ethanol, M9-N buffer, conducted at 22 °C under aerobic condition for 18 h. Ptotal, sum area for all diastereomers. Grey bars indicate the dr. Data are mean ± s.d.; n = 3 reaction replicates.
Extended Data Fig. 6 CYP203A1 WT and axial ligand mutants for the reaction of styrene with azaserine.
EIC ([M+H]+, m/z 250.1074) for target products. Representative traces for two repeated experiments. The reactions contained 5 mM styrene, 5 mM azaserine, E. coli cells with concentration of 30 OD600 as catalyst, 5 vol% ethanol, and M9-N buffer and were conducted at 22 °C under aerobic conditions for 18 h.
Extended Data Fig. 7 Conserved amino acids selected for saturation mutagenesis.
The residue labelled in blue is the haem ligand cysteine in those P450s. Residues labelled in red are two conserved residues previously reported to affect the catalytic behaviour in P450 BM3. Sequences were aligned with Clustal Omega64.
Extended Data Fig. 8 Exploration of the conditions for styrene biosynthesis in S. albus.
a, Heterologously expressing PAL2 and FDC1 are sufficient to generate styrene. pAZA121 and pAZA138 are two integration plasmids used to introduce the styrene pathway into S. albus. b, Production of styrene by engineered S. albus grown in TSB medium supplemented with additional 0 mM, 2 mM or 4 mM Phe. Data are mean ± s.d.; n = 3 biological samples.
Extended Data Fig. 9 Characterization and time course of accumulation of the final products.
a, MS/MS (20 eV) spectra of the P1 standard (red) and the biosynthesized major product (black). b, Biosynthesis of final products during the 96-h fermentation process. The 24-h data are not presented because small quantities of product were observed (<10 µg/L), and thus the titre could not be accurately calculated. 1B medium with 4 mM Phe was used to generate the final products. Data are mean ± s.d.; n = 3 biological samples.
Supplementary Tables 1–3 (DNA and proteins sequences, synthesis of compounds), Supplementary Figs. 1–38 and Supplementary References.
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Huang, J., Quest, A., Cruz-Morales, P. et al. Complete integration of carbene-transfer chemistry into biosynthesis. Nature 617, 403–408 (2023). https://doi.org/10.1038/s41586-023-06027-2
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